Most bacterial pathogens have an absolute requirement for iron. The low availability of iron in most environments has led to the evolution of high affinity iron transport systems. Although a variety of iron transport systems have been identified in Shigella and pathogenic E. coli, the sources of iron used by the pathogens when growing within the host and the specific iron transport systems involved in growth and survival in vivo are not known. Shigella species, the causative agents of dysentery, are closely related to E. coli and share many of the same iron transport systems. However, differences have been noted among this group of pathogens and those differences may relate to differences in sources of iron at various sites within the host or in the environment. Because Shigella spp. have the ability to invade host cells and grow with the cytosol, there may be specific iron transport systems associated with iron acquisition in the intracellular environment. Our first specific aim is to complete the characterization of the Shigella heme transport systems. Many shigellae and pathogenic E. coli, including O157:H7, have specific receptors for heme. Although receptors and other proteins involved in transporting heme across the bacterial cell wall have been identified, other steps in heme acquisition and its use as an iron source are not understood. The second aim is to use genetic and genomic approaches to identify the additional iron uptake systems in these pathogens. We will then apply these data and use the mutants created in these studies to help understand the role of the iron transport systems in growth and survival in vivo and in the environment. Thus our third specific aim is to determine which systems are used under specific environmental conditions and during intracellular growth. Our fourth specific aim is to assess expression of iron transport and other genes during intracellular growth and during infection by using microarray technology.